Project Summary/Abstract This proposed training plan will provide high-quality professional development for the applicant through the pursuit of experiments designed to determine the impact of chronic exercise, physical inactivity, and cardiovascular disease on mechanisms that mediate endothelial cell function, with the goals of addressing a critical barrier to our current understanding of cardiovascular disease and eventual application of these mechanisms towards the clinical treatment and prevention of cardiovascular disease. The isoenzyme 6- phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) regulates glycolytic metabolism and is essential for endothelial cell function. Understanding the role of PFKFB3 in endothelial cell metabolism and function has recently emerged, and as a result the current literature merely describes the effect of cardiovascular disease on perturbations in endothelial cell metabolism and function. Research identifying the mechanisms underlying the development of cardiovascular disease-induced deregulations in endothelial cell metabolism and function are non-existent. Indeed, a lack of chronic exercise (physical inactivity) reduces endothelial function and increases the risk for cardiovascular disease, whereas chronic exercise training ameliorates cardiovascular disease risk and enhances vascular function. The possibility that chronic exercise may confer protection against cardiovascular disease through functional adaptations in endothelial cell metabolism has not been addressed. Therefore, the overall goal of the present application, in combination with carefully integrated career development activities and mentors with expertise in biology, cell metabolism, and exercise physiology, is to describe the extent to which endothelial metabolism mediates the function of endothelial cells across a spectrum of cardiovascular disease risk, from the atheroprotective effects conferred through chronic exercise at one end, to the physical inactivity-induced manifestation of endothelial dysfunction in the middle, to overt cardiovascular disease on the other end as an extreme. Serum samples collected from individuals who perform chronic exercise, physically inactive but healthy participants, and patients with overt cardiovascular disease will be cultured on human umbilical vein endothelial cells to isolate the effect of circulating factors on endothelial cell metabolism and function. In the final set of experiments, serum from the aforementioned populations will be cultured on human umbilical vein endothelial cells with PFKFB3 either overexpressed or knocked down to determine how alterations in endothelial cell metabolism mediate endothelial cell function across the spectrum of cardiovascular disease risk. Identifying the role of endothelial metabolism in vascular homeostasis will have far broader implications than exercise alone, with the results of the proposed experiments addressing a critical barrier to our current understanding of cardiovascular disease and eventual application of these mechanisms to viable therapeutic interventions designed to ameliorate and prevent cardiovascular disease.